CN106755046A - A kind of method for transforming bacillus gene group - Google Patents
A kind of method for transforming bacillus gene group Download PDFInfo
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Abstract
The invention belongs to biological technical field, and in particular to a kind of method of transformation bacillus gene group.The present invention has wide adaptability, and (plasmid conversion difficulty is low,PBTS plasmids can be replicated in various gram-positive bacterias,Transform the genome of various bacterium),Can (recombinant plasmid has two homologous recombination fragments with the same bacterial strain of repeated multiple times transformation,There is homologous recombination twice successively,Wild type may be returned to after second homologous recombination,Can also obtain transforming bacterial strain,But the fragment that any influence continues to transform is not remained,Such as resistance marker,Plasmid replication element etc.),And can transform influences larger gene (to introduce cre recombinases and second resistance marker system on bacterial growth,The transformation bacterial strain for obtaining can be for the second time recombinated by resistance screening,Kill the wild-type strain of survival ability is strong,Cre recombinases are expressed afterwards,Cut away second resistance marker,The fragment of residual is not interfered with to be transformed bacterial strain again).
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of method of transformation bacillus gene group.
Background technology
Protein expression techniques are one of core technologies of modern biology, and expressing protein cannot be only used for biological study,
Commercialized protein product, such as recombinant vaccine, Recombulin, cell factor product can also be provided.The table commonly used at present
There are Escherichia coli, yeast, insect cell and mammalian cell etc. up to system, but they all respectively have obvious advantage and disadvantage.Greatly
The research of enterobacteria expression system most fully, there is a multiple choices, the pET expression systems of most commonly Novagen, and it utilizes phagocytosis
The t7 rna polymerase of body can in specific manner transcribe the genes of interest after T7 promoters.At optimum conditions, destination protein can reach
To more than the 50% of e. coli total protein.Although having expression efficiency high, the low advantage of toxigenic capacity, shortcoming is also very
Substantially：Albumen easily forms inclusion body, renaturation difficulty and relatively costly；Escherichia coli can not carry out glycosylation modified to albumen；
Cell membrane contains lipopolysaccharides (endotoxin), is difficult to remove completely.Another conventional expression system is yeast expression system, its
It is high with expression quantity, can induce, PE is easy to purifying to extracellular, and excellent with certain posttranslational modification ability etc.
Point, but a disadvantage is that part expression product is degradable, expression quantity is uncontrollable, and the albumen more than 30KDa can hardly be secreted.Animal
The characteristics of cell and insect cell expression system is that, with complete modification system, expression product has or similar natural work
Property, there is no contaminated with endotoxins, but expression quantity is low, and the cycle is long, and technical requirements are high, and production cost is high.
Bacillus subtilis is a kind of gram-positive bacteria being widely present in water body, air, soil, and it can be in ring
Spore, spore are produced when border is unfavorable can resist the extreme environments such as high temperature, arid, and being sprouted again when environment is suitable is carried out
Nutrient growth.The secretion capacity of bacillus subtilis is strong, and when high density fermentation is carried out, protein secretion can reach 20-
25g/L.The products such as protease, amylase, inosine, the riboside of its fermenting and producing, already into our day
Often life.Because it has biological safety, by FDA judging panel's GRAS class additives, the probiotics of its production and using its production
Fermented product be widely used to medicine and aquaculture in.
Last century the eighties begin to carry out protein expression using bacillus subtilis, and its expression product not only includes thin
The various enzymes in bacterium source, such as amylase, protease, endo-dextranase, lipase, also including hEGF, IFN-alpha 2,
The albumen of the separate sources such as Proinsulin, Streptavidin, cathelicidin-BF.Using dividing for bacillus subtilis
Approach is secreted, expressing protein can be secreted into zymotic fluid, significantly reduce the difficulty that the later stage isolates and purifies.Bacillus subtilis
Belong to gram-positive bacteria, do not contain lipopolysaccharides, be easy to produce injection medicine.The nutritional requirement of bacillus subtilis is simple,
Produced because it has been carried out technical scale metaplasia, large-scale culture technical difficulty is low, and toxigenic capacity is low.At present, bacillus subtilis
Multiple kind in multiple bacterial strains and bacillus in bacterium all has been completed that gene order-checking works, and genetic background understands,
It is safe, also allow for carrying out genome manipulation.But bacillus subtilis expression system is not a wide variety of table
Up to system, also many shortcomings are to be overcome.
The wild-type strain of the bacterium of pattern bacterium 168 of bacillus subtilis has been lost, and the bacterial strain being currently available all is
Its mutant.Although it is satisfied with the requirement of scientific research, its PE ability, is auxotrophic mutations
Characteristic, can not meet the requirement for setting up commercialization expression system.The conversion system set up at present is all based on type strain 168
Set up, although the bacterial strain in wild type source and the bacillus subtilis strain of production application are very more, are difficult conversion and all hinder
The further transformation to these bacterial strains is hindered.The plasmid in bacillus subtilis source is also nearly all stealthy plasmid, is difficult to build
Carrier；All it is rolling-circle replication property grain from the plasmid majority of other gram-positive bacterias, is replicated in bacillus subtilis
Unstable, easy to lose, copy number is less；These characteristics all limit possibility directly using plasmid construction stabilization expression system
Property.Bacillus subtilis has four kinds of secretory pathways, Sec approach, Tat approach, Com systems and abc transport approach, research at present compared with
Sec approach is clear that, some expression systems also utilize Sec approach secreting, expressing albumen.But Sec approach has regulation and control machine
System, can suppress the PE of incorrect folding, and foreign protein is typically folded relatively slowly, and folding process needs the ginseng of chaperone
With easily degraded by Sec secretory pathways.Bacillus subtilis can also secrete up to eight kinds protease in zymotic fluid, and its is same
Can be degraded the destination protein of expression.
Overcoming the effective way of disadvantages mentioned above is：Plasmid is not used as genes of interest and the carrier of expression system element,
Directly by genes of interest and expression system element insertion Bacillus subtilis genes group；Its genome is transformed, is knocked out
Some suppress or influence the gene of PE.The method of current transformed bacillus bacillus gene group has two kinds, the first
It is directly to convert double chain DNA fragment into cell while carrying out homologous recombination, it is high to transformation efficiency requirement, is currently only used for
168 pattern bacterium.Second is to utilize temperature-sensitive plasmid, and temperature-sensitive plasmid of the conversion with homologous recombination fragment enters thin
Born of the same parents, then cultivate under restrictive temperature, there occurs the bacterium of restructuring and the bacterium of homologous recombination can not occur due to matter by screening
Grain is lost, it is impossible to by screening.The conversion of the method and reconstitution steps are separated, and reduce the requirement to transformation efficiency.Can select
The temperature-sensitive plasmid selected is pE194, but the plasmid is a plasmid that can only be replicated in gram-positive bacteria, it is impossible to
Replicated in Escherichia coli, i.e., can not quickly be carried out plasmid construction work using Escherichia coli.One of solution is structure
The fusion plasmid of fusion plasmid, i.e. pE194 and pBR322 is built, the plasmid possesses two sets of dubbing systems, and difference can be in withered grass bud
Replicated in spore bacillus and Escherichia coli.But stability is poor when the plasmid is replicated in Escherichia coli, if polyclonal
When site insertion is less than the fragment of 1.5k, plasmid can stablize duplication；If Insert Fragment be more than 2k, the plasmid replicate when just
The phenomenon of fragment loss can occur.Another conventional temperature-sensitive plasmid is pKS1, and the plasmid is host range plasmid more than, can
Replicated with most gram-positive bacterias and negative bacterium, but the plasmid stability is poor, and copy number is relatively low.Document
(Rational Design of a Plasmid Origin That Replicates Efficiently in Both
Gram-Positive and Gram-Negative Bacteria, 2010) the original plasmid pWV01 of pKS1 is transformed,
Its stability and copy number in Escherichia coli is increased, vector construction is convenient for.But not to transformation plasmid in text
The temperature-sensing property of pBAV1K is studied, and temperature of the pBAV1K-T5-GFP in bacillus subtilis is found in our work
Degree sensitive copy characteristic is still present, but restricted cultivation temperature is increased to 45 DEG C from 37 DEG C of pKS1.According to pBAV1K-
These characteristics of T5-GFP plasmids, the method that a set of transformed bacillus bacillus gene group can be developed.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of method for transforming bacillus gene group, using many places
Main high copy number plasmid pBAV1K stability in Escherichia coli is high, and copy number is more, it is easy to the characteristic of transformation and in bacillus
Temperature sensitive copy characteristic, the method for constructing a set of transformed bacillus bacillus gene group, with wide adaptability, transformation
Plasmid is easy to conversion, can be replicated in various bacillus, transforms various bacillus；Can repeat to transform same bacterium
Strain, the fragment of influence transformation next time such as resistance, reproduction element is not remained every time after transformation；Can also transform influences on strain growth
Larger gene, introduces cre recombinases and the second resistance screening mark.
The method for solving a kind of transformation bacillus gene group in the present invention of above technical problem, it is characterised in that：
Comprise the following steps：
(1) EcoR I and the Apa I site in pBAV1K-T5-GFP plasmids insert MCS (MCS) fragment, and
By same sense mutation, NdeI restriction enzyme sites therein are deleted, obtain plasmid pBTS.
PBTS plasmids can be replicated in various gram-positive bacterias, transform the genome of various bacterium.
(2) restriction enzyme site in the left end in MCS sites inserts the homologous recombination fragment A of 500-800bp, right side restriction enzyme site
The homologous recombination fragment B of 500-800bp is inserted, middle insertion mutation fragment or purpose fragment or any fragment is not inserted into, both
The fragment of transformation is needed between two homologous recombination regions, plasmid pBTS-X is obtained.
Here left end and the restriction enzyme site of right-hand member can be any restriction enzyme site, it is only necessary to ensure A before B just；
A, B represent two sections of sequences respectively, specifically may refer to Technology Roadmap, and A, B are any two sections of sequences；Mutant fragments, purpose piece
Section can be any fragment, or you think the fragment of transformation, are only represented with X here, referring specifically to Technology Roadmap or implementation
Example.
(3) pBTS-X is transferred in bacillus by electricity conversion, obtains positive transformants bacterial strain；
(4) into being cultivated in fluid nutrient medium, cultivation temperature is 45 DEG C to picking positive transformants bacterium colony, and incubation time is big
In 24h.
(5) bacterium solution 100ul -500ul, is applied on the flat board containing kanamycins in taking 4, and cultivation temperature is 45 DEG C, is entered
Row incubated overnight.
(6) bacterium colony in picking 5 is into being cultivated in fluid nutrient medium, every 8-16h passages once, until screening
Nonreactive bacterial strain.I.e. same bacterial strain, does not grow on containing resistant flat board, is grown on the flat board of nonreactive.
(7) take the bacterium solution in 6 and be diluted coated plate, dilution 105-106Times, incubated overnight.
(8) bacterium colony in picking 7 carries out Resistance Identification, until obtaining 5-10 plants of nonreactive bacterial strain.
(9) nonreactive bacterial strain taken in 8 is inoculated into fluid nutrient medium, incubated overnight, extracts bacterial genomes, enters performing PCR
Identification；Positive strain proceeds sequencing identification, and sequencing identification result is the bacterial strain for completing genome manipulation for positive strain.
In the step (3) bacillus be bacillus subtilis 168, Z12 bacterial strains, also including other bacillus, such as
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus pumilus (Bacillus pumilus),
The bacterial strains such as clothing bacillus (Bacillus licheniformis), or other pBTS plasmids have temperature sensitive copy characteristic
Gram-positive bacteria.
The gram-positive bacteria is streptococcus pneumonia (Streptococcus pneumoniae), Lactococcus lactis
(Lactococcus lactis) or series bacillus (Paenibacillus sp) bacterial strain.
The pBTS nucleotide sequences are as shown in SEQ ID NO.1.
For in 6-9 steps in the present invention, second restructuring can obtain wild-type strain and transformation bacterial strain, if wild
The growth ability of type bacterial strain is better than transformation bacterial strain, is difficult to screen the situation of transformation bacterial strain, the strain improvement scheme of design optimization,
Step is as follows：
(1) the MCS sites left-hand end in pBTS inserts the homologous recombination fragment A of 500-800bp, right-hand end insertion 500-
The homologous recombination fragment B of 800bp, middle insertion mutation fragment or purpose fragment or is not inserted into fragment, then in the transformation of B with centre
Insertion has blasticidin resistance fragment between fragment or A and the transformation fragment of centre, and blasticidin resistance fragment two ends contain
There are lox71 the and lox72 fragments that cre recombinases are recognized, the plasmid is named as pBTS-X-Z.Resistance fragments herein are not limited only to
Blasticidin resistance fragment, also resistance fragments such as including similar tetracycline, chloramphenicol, spectinomycin.
(2) pBTS-X-Z plasmids are converted by electricity conversion and is entered in bacillus, bacillus herein includes withered grass
The bacterial strains such as bacillus 168, Z12, also including other bacillus, such as bacillus amyloliquefaciens, bacillus pumilus, lichens bud
The bacterial strains such as spore bacillus, such as gram-positive bacteria also including other pBTS plasmids with temperature sensitive copy characteristic, pneumonia streptococcus
Bacterium (Streptococcus pneumoniae), Lactococcus lactis (Lactococcus lactis), series bacillus
Bacterial strains such as (Paenibacillus sp.).
(3) into being cultivated in fluid nutrient medium, cultivation temperature is 45 DEG C to the positive transformants bacterium colony in picking (1), training
The time of supporting is more than 24h.
(4) bacterium solution 100ul -500ul in (3) is taken, is applied on the flat board containing kanamycins or bleomycin, trained
It is 45 DEG C, incubated overnight to support temperature.
(5) bacterium colony in picking (4) enters and is cultivated in the LB liquid medium containing blasticidin resistance, every 8-
16h is passed on once, the bacterium colony only until screening with bleomycin.
(6) take the bacterium solution in (5) and be diluted coated plate, flat board contains blasticidin resistance, dilution 105-106Times, overnight train
Support.
(7) bacterium colony in picking (6) carries out Resistance Identification, only has blasticidin resistance until obtaining 3-5, without card
The bacterial strain of that chloramphenicol resistance.
(8) monoclonal antibody inoculation in (7) is taken in culture medium, and incubated overnight extracts bacterial genomes, enters performing PCR mirror
It is fixed.Positive strain proceeds sequencing identification, and the positive strain of sequencing identification enters next step.
(9) conversion pBTS-IC plasmids enter the monoclonal antibody bacterial strain that the positive is accredited as in (8), and IC is that lactose operator regulates and controls cre
Recombinate the fragment of expression of enzymes, cre recombinases can characteristic identification lox71 and lox66 sites, recombinated, left in genome
The loxP sites that can not be recognized by cre recombinases, the lower fragment of cutting contains the lox72 sites for easily recognizing.
(10) the positive transformants bacterial strain in picking (9), connects bacterium and is cultivated, using IPTG induced expressions or leakage expression
Cre restructuring cleavage blasticidin resistance fragment.
(11) bacterium solution in (10) is taken, is inoculated into fluid nutrient medium, cultivation temperature is 45 DEG C, and incubation time is more than 24h.
(12) bacterium solution of culture in (11), dilution 10 are taken5-106To on the flat board of nonreactive, cultivation temperature is 45 DEG C, mistake to coated plate
Night cultivates；
(13) single bacterium colony in (12) is taken, Resistance Identification is carried out, until screens 2-5 plants of nonreactive bacterial strain.
(14) nonreactive bacterial strain in (13) is taken, is inoculated into culture medium, incubated overnight extracts genome, enter performing PCR mirror
It is fixed.Positive strain carries out sequencing identification again, and the bacterial strain for being still accredited as the positive is the bacterial strain for completing genome manipulation.
The pBTS-IC nucleotide sequences are as shown in SEQ ID NO.3.
The skeleton of pBTS plasmids is that stability is high, the pBAV1K plasmids more than copy number, multiple relative to other responsive to temperature type
Plasmid processed, it has dubbing system element fragment short (comprise only a kind of dubbing system element and kalamycin resistance is marked), can
Replicated with Escherichia coli and gram-positive bacteria, duplicate copy number is high, stability it is high (fragment of insertion 6K or so,
The imagination of fragment loss will not also occur).
The method that the homologous recombination based on pBTS transforms bacterial genomes in the present invention, with wide adaptability, (plasmid is converted
Difficulty is low, and pBTS plasmids can be replicated in various gram-positive bacterias), can be with the repeated multiple times same bacterial strain of transformation (weight
Group plasmid has two homologous recombination fragments, and homologous recombination twice occurs successively, and open country may be returned to after second homologous recombination
Raw type, it is also possible to obtain transforming bacterial strain, but do not remain the fragment that any influence continues to transform, such as resistance marker, plasmid replication
Element etc.), and can transform influences larger bacterial strain (to introduce cre recombinases and second resistance marker system on bacterial growth
System, can for the second time recombinate the transformation bacterial strain for obtaining by resistance screening, kill the wild-type strain of survival ability is strong, express afterwards
Cre recombinases, cut away second resistance marker, and the fragment of residual is not interfered with to be transformed bacterial strain again).
Brief description of the drawings
Fig. 1 is Technology Roadmap in the present invention
Fig. 2 is the Technology Roadmap of optimization in the present invention
Fig. 3 is the pBTS structure charts in the present invention
Fig. 4 is the pBTS-IC structure charts in the present invention
Specific embodiment
With reference to specific embodiment, the present invention is described in further detail:
Embodiment 1
(1) by EcoR I and Apa I inscribe cleavage pBAV1K-T5-GFP plasmids, the MCS fragments with synthesis are carried out together
Source recombinates, and obtains plasmid pBAV1K.
Restriction endonuclease in this experiment and follow-up test uses the quick restriction endonuclease of Thermo companies, and fragment is reclaimed and uses Chengdu
The glue reclaim kit (DE-02011) of Fu Ji biotech firms, MCS fragments are the synthesis of purchase Jin Wei intelligence bio tech ltd
Fragment, homologous recombination using Tiangeng EsayGeno Quick Castings Cloning Kit (VI201-02), coli strain is
Top10, prepares competence and uses KCM methods, and plasmid extraction is using the biological universal plasmid Mini Kit (DE- in good fortune border
01001)。
(2) primer is designed, the pBAV1K fragments of Nde I restriction enzyme sites are deleted in amplification same sense mutation, using homologous recombination gram
Grand mode junction fragment, obtains plasmid and is named as pBTS.
Primer in this experiment and subsequent experimental is synthesized by Jin Wei intelligence bio tech ltd.
Embodiment 2：
Full genome synthesizes IC fragments, is inserted between XbaI the and Pst I of pBTS by homologous recombination, obtains plasmid
pBTS-IC.Here what is obtained is the pBTS-IC plasmids in preferred scheme；PBTS-FR-X may refer to following transformation xylAB
The plasmid of gene.
Embodiment 3：
1st, the fragment xyl-F of the xylose xylAB operators upstream 493bp of design primer amplification Z12 bacterial strains, insertion pBTS's
Between EcoR I and BamHI sites, plasmid pBTS-xyl-F is obtained；Expand the fragment xyl-R of downstream 620bp, insertion plasmid pBTS-
Between BamHI the and Hind III of xyl-F, plasmid pBTS-xyl plasmids (knocking out xylAB operators) are obtained.High-fidelity enzyme is used
KOD-Plus hi-fis polymerase (KOD-201) of toyobo.
2nd, design primer expand Z12 bacterial strains pgsBCA operators upstream 693bp fragments, insert pBTS EcoR I and
Between BamHI sites, obtain plasmid pBTS-pgs-F amplification downstream 558bp fragments, the BamHI of insertion plasmid pBTS-pgs-F and
Between Hind III, plasmid pBTS-pgs is obtained.Full genome synthesizes zeo fragments, inserts the BamHI sites of pBTS-pgs, obtains
Plasmid pBTS-pgs-Z (knocks out pgsBCA operators, but contains blasticidin resistance mark in the middle of homologous recombination fragment).
Embodiment 4：
Bacterial strain method for transformation is referring to hypertonic conversion method (High osmolarity improves the electro-
transformation efficiency of the gram-positive bacteria Bacillus subtilis and
Bacillus licheniformis, 1999).The new line single bacterium colony of Z12 bacterial strains is taken, about 3ml GM (LB+0.5M mountains are inoculated into
Pears sugar alcohol) in culture medium, 37 DEG C, 180rpm incubated overnights.The next morning presses 1:100 are inoculated into 50ml GM culture mediums, and 37
DEG C, bacterium solution when bacterium solution grows into OD and reaches between 0.85-0.95, is put into precooling 10min on ice by 180rpm cultures；4 DEG C,
Supernatant is removed in 5000g, 5min, centrifugation, and with the EM of isometric precooling, (0.5M D-sorbite+0.5M+10% glycerine of mannitol is water-soluble
Liquid) resuspended thalline, 4 DEG C again, supernatant is removed in 5000g, 5min, centrifugation, repeated washing 4 times altogether；Add the EM of about 1/40 volume
Resuspended thalline, it is ensured that bacterial concentration is in 1-1.3 × 1010Between cfu/ml.The above-mentioned bacterium solutions of 60ul are taken, 1ul plasmids to be transformed are added
(pBTS-xyl and pBTS-pgs-Z), plasmid concentration is more than 100ng/ul, and piping and druming is mixed, and is subsequently adding the 1mm electric shock cups of precooling.
Electric conversion instrument (Eppendorf Eporator) parameter setting, 2.1kV, the actual electric shock time, in 4.0-5.0ms, has been only possible to
Conversion colony growth.After electric shock.Immediately plus 1ml RM (LB+0.5M D-sorbite+0.38M mannitols), piping and druming is mixed, and is transferred to
In 5ml sterile centrifugation tubes, 37 DEG C, 180rpm cultures 3h.Bacterium solution is centrifuged, is diluted by a certain percentage after removing supernatant, take 200ul paintings
Cloth on the LB flat boards containing 30mg/L kanamycins, 37 DEG C of incubated overnights, while bacterium solution of the coating without electricity conversion does feminine gender
Control.The next morning observes colony growth situation, if reformer plate has colony growth, negative control does not have, then show to change into
Work(.It is Z12-pBTS-xyl or Z12-pBTS-pgs-Z to name the bacterial strain for obtaining.
Embodiment 5：
(1) take bacterium Z12-pBTS-xyl to be inoculated into 3ml LB culture mediums, 45 DEG C, 180rpm, cultivate 24h；Separately connect bacterium
Z12-pBTS is compareed.
(2) take the bacterium solution 200ul in 1 to be applied on the LB flat boards containing 30mg/L kanamycins, 45 DEG C of incubated overnights.If
Z12-pBTS does not have colony growth, and Z12-pBTS-xyl has colony growth, then the bacterial strain for obtaining completes to recombinate for the first time, is named as
Z12-pBTS-xyl-45。
(3) connect in bacterium Z12-pBTS-xyl-45 to 3ml LB culture mediums, incubated overnight, extracting genome (is given birth on Chengdu good fortune border
Thing Technology Co., Ltd., bacterial genomes DNA extraction kit, DE-05311), PCR amplifications xyl fragments are verified (Chengdu
Fu Ji Bioisystech Co., Ltd, 2 × fast PCR reaction premix system, DP-20041.Primer sequence, F：
CAAAATGTCTTTCGTTATTTCTGGAG, R：AGTGTTCGCTGACAAAATACGGTTC, 60 DEG C of annealing, 2min extends).
(4) the Z12-pBTS-xyl-45 bacterial strains that the positive is verified as in 3 are taken, is inoculated into 37 DEG C in 3ml LB bases, 180rpm trainings
Support, every 8-16h passages once, after continuous passage 8 times, take bacterium solution dilution 106Times, coat on the LB flat boards of nonreactive, obtain
Single bacterium colony.
(5) single bacterium colony in 4 is taken, while being scoring on LB and the LB flat boards containing 30mg/L kanamycins, nonreactive is screened
Bacterium colony, it is necessary to obtain 5-10 plants of nonreactive bacterium colony.
(6) the nonreactive bacterium colony in 5 is taken, is inoculated into 3ml LB culture mediums, 37 DEG C, 180rpm incubated overnights, extract bacterium
Genome, expands xyl fragments and verifies (Chengdu Fu Ji Bioisystech Co., Ltd, 2 × fast PCR reaction premixed material by PCR
System, DP-20041.Primer sequence, F：CAAAATGTCTTTCGTTATTTCTGGAG, R：
AGTGTTCGCTGACAAAATACGGTTC, 60 DEG C of annealing, 2min extends) (wild type knocks out xyl bases to homologous recombination result
Cause).The fragment of the positive is taken, Song Jinwei intelligence bio tech ltd further carries out sequence verification, obtain the bacterial strain of the positive i.e.
To knock out bacterial strain --- the Z12 (△ xylAB) of xyl genes.
(7) Z12 (△ xylAB) and Z12 bacterial strains are taken, according to the wood-sugar fermentation experimental verification in primary Jie Shi Bacteria Identifications handbook
The ability of its fermenting xylose, as a result shows that Z12 can be with fermenting xylose, but Z12 (△ xylAB) is unable to fermenting xylose.
Embodiment 6：
(1) take bacterium Z12-pBTS-pgs-Z to be inoculated into 3ml LB culture mediums, 45 DEG C, 180rpm, cultivate 24h；Separately connect bacterium
Z12-pBTS is compareed.
(2) take the bacterium solution 200ul in 1 to be applied on the LB flat boards containing 30mg/L kanamycins, 45 DEG C of incubated overnights.If
Z12-pBTS does not have colony growth, and Z12-pBTS-pgs-Z has colony growth, then the bacterial strain for obtaining completes to recombinate for the first time, name
It is Z12-pBTS-pgs-Z-45.
(3) connect in bacterium Z12-pBTS-xyl-45 to 3ml LB culture mediums, incubated overnight extracts genome, PCR amplifications pgs
Fragment is verified (primer sequence, F：AGCTGTTCCGATTTTATACTGGTC, R：TAGCCAAAACAGCTCCTCCTTG, 60
DEG C annealing, 2min extend).
(4) the Z12-pBTS-pgs-Z-45 bacterial strains that the positive is verified as in 3 are taken, is inoculated into and is contained 30mg/L bleomycins
37 DEG C in 3mlLB bases, 180rpm cultures every 8-16h passages once, after continuous passage 10 times, take bacterium solution dilution 106Times, apply
It is distributed on the LB flat boards containing 30mg/L bleomycins, obtains single bacterium colony.
(5) single bacterium colony in 4 is taken, while being scoring to containing 30mg/L kanamycins and containing 30mg/L bleomycins
On LB flat boards, screening comprises only the bacterium colony of blasticidin resistance, it is necessary to obtain 3-5 plants of monoclonal antibody bacterium colony.
(6) the monoclonal antibody bacterium colony in 5 is taken, is inoculated into 3ml LB culture mediums, 37 DEG C, 180rpm incubated overnights, extract bacterium
Genome, expands pgs fragments and verifies (primer sequence, F by PCR：AGCTGTTCCGATTTTATACTGGTC, R：
TAGCCAAAACAGCTCCTCCTTG, 60 DEG C of annealing, 2min extends) homologous recombination result (wild type knocks out pgs genes).
It is Z12-pgs-Z to name the positive strain.
(7) Z12-pgs-Z bacterial strains are entered by electricity conversion pBTS-IC plasmids, is named as Z12-pgs-Z-pBTS-IC.
(8) bacterium Z12-pgs-Z-pBTS-IC is met in the 3ml LB culture mediums containing 1mM IPTG, 37 DEG C, 180rpm, mistake
Night cultivates.
(9) take the bacterium solution in 8 to be inoculated into 3ml LB culture mediums, 45 DEG C, 180rpm, cultivate 24h.Bacterium solution dilution 106Times,
It is applied on the LB flat boards of nonreactive, 45 DEG C, incubated overnight obtains single bacterium colony.
(10) take the single bacterium colony in 9 and contain 30mg/L kanamycins, 30mg/L bleomycins and nonreactive while being scoring to
On LB flat boards, the bacterial strain of nonreactive is screened.
(11) nonreactive bacterial strain in 10 is taken, is inoculated into 3ml LB culture mediums, 37 DEG C, 180rpm, incubated overnight.Extracting base
Because of group, pgs fragments are expanded by PCR and verifies whether correctly to cut blasticidin resistance fragment.To positive fragment, Jin Weizhi is sent
Bio tech ltd carries out sequencing identification, knocks out pgsBCA operators and does not contain the bacterial strain of blasticidin resistance mark i.e.
To complete the bacterial strain of transformation, Z12 (△ pgsBCA) is named as.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Sequence table
SEQUENCE LISTING
<110>Chengdu Mei Yide Bioisystech Co., Ltd
<120>A kind of method for transforming bacillus gene group
<160>3
<170>PatentIn version 3.3
<210>1
<211>2847
<212>DNA
<213>It is artificial synthesized
<220>
<223>PBTS sequences
<400>1
gacgtcgaattcgaggtacctgcggccgcaggatccatctagacgctcgagagctgcagtgaagcttcagggcccga
tcgatgccgccgcttaattaattaatccagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcg
ttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctagtgtcattttattt
cccccgtttcagcatcaagaacctttgcataacttgctctatatccacactgataattgccctcaaaccataatcta
aaggcgctagagtttgttgaaacaatatcttttacatcattcgtatttaaaattccaaactccgctcccctaaggcg
aataaaagccattaaatcttttgtatttaccaaattatagtcatccactatatctaagagtaaattcttcaattctc
ttttttggctttcatcaagtgttatatagcggtcaatatcaaaatcattaatgttcaaaatatcttttttgtcgtat
atatgtttattcttagcaatagcgtcctttgattcatgagtcaaatattcataagaacctttgatataatcaagtat
ctcaacatgagcaactgaactattccccaattttcgcttaatcttgttcctaacgctttctattgttacaggatttc
gtgcaatatatataacgtgatagtgtggttttttatagtgctttccatttcgtataacatcactactattccatgta
tctttatcttttttttcgtccatatcgtgtaaaggactgacagccatagatacgcccaaactctctaatttttcctt
ccaatcattaggaattgagtcaggatataataaaaatccaaaatttctagctttagtatttttaatagccatgatat
aattaccttatcaaaaacaagtagcgaaaactcgtatccttctaaaaacgcgagctttcgcttattttttttgttct
gattcctttcttgcatattcttctatagctaacgccgcaaccgcagattttgaaaaacctttttgtttcgccatatc
tgttaattttttatcttgctcttttgtcagagaaatcataactctttttttcgattctgaaatcaccatttaaaaaa
ctccaatcaaataattttataaagttagtgtatcactttgtaatcataaaaacaacaataaagctacttaaatatag
atttataaaaaacgttggcgaaaacgttggcgattcgttggcgattgaaaaaccccttaaacccttgagccagttgg
gatagagcgtttttggcacaaaaattggcactcggcacttaatggggggtcgtagtacggaagcaaaattcgcttcc
tttccccccatttttttccaaattccaaatttttttcaaaaattttccagcgctaccgctcggcaaaattgcaagca
atttttaaaatcaaacccatgagggaatttcattccctcatactcccttgagcctcctccaaccgaaatagaagggc
gctgcgcttattatttcattcagtcatcggctttcataatctaacagacaacatcttcgctgcaaagccacgctacg
ctcaagggcttttacgctacgataacgcctgttttaacgattatgccgataactaaacgaaataaacgctaaaacgt
ctcagaaacgattttgagacgttttaataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccggcgg
caaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagct
cggtactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaa
aaatagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgt
ccgccctgccgcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccagg
tcgccgtgggaaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaa
tggagtgtcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagc
ggccgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagc
tcgatagtcttttcagggctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatgagcag
attgctccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgt
ccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggatttcattttct
cccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatcagtttttt
caattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaaga
agctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaacagccttttc
aaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgatagaattt
2847
<210>2
<211>3344
<212>DNA
<213>It is artificial synthesized
<220>
<223>PBTS-Z sequences
<400>2
gacgtcgaattcgaggtacctgcggccgcaggatcctaccgttcgtatagcatacattatacgaagttatcttgata
tggctttttatatgtgttactctacatacagaaaggaggaactaaatatggccaagttgaccagtgccgttccggtg
ctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggagga
cgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaaca
ccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgg
gacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccgg
caactgcgtgcacttcgtggccgaggagcaggactgaataacttcgtatagcatacattatacgaacggtaGtctag
acgctcgagagctgcagtgaagcttcagggcccgatcgatgccgccgcttaattaattaatccagaggcatcaaata
aaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggac
aaatccgccgccctagacctagtgtcattttatttcccccgtttcagcatcaagaacctttgcataacttgctctat
atccacactgataattgccctcaaaccataatctaaaggcgctagagtttgttgaaacaatatcttttacatcattc
gtatttaaaattccaaactccgctcccctaaggcgaataaaagccattaaatcttttgtatttaccaaattatagtc
atccactatatctaagagtaaattcttcaattctcttttttggctttcatcaagtgttatatagcggtcaatatcaa
aatcattaatgttcaaaatatcttttttgtcgtatatatgtttattcttagcaatagcgtcctttgattcatgagtc
aaatattcataagaacctttgatataatcaagtatctcaacatgagcaactgaactattccccaattttcgcttaat
cttgttcctaacgctttctattgttacaggatttcgtgcaatatatataacgtgatagtgtggttttttatagtgct
ttccatttcgtataacatcactactattccatgtatctttatcttttttttcgtccatatcgtgtaaaggactgaca
gccatagatacgcccaaactctctaatttttccttccaatcattaggaattgagtcaggatataataaaaatccaaa
atttctagctttagtatttttaatagccatgatataattaccttatcaaaaacaagtagcgaaaactcgtatccttc
taaaaacgcgagctttcgcttattttttttgttctgattcctttcttgcatattcttctatagctaacgccgcaacc
gcagattttgaaaaacctttttgtttcgccatatctgttaattttttatcttgctcttttgtcagagaaatcataac
tctttttttcgattctgaaatcaccatttaaaaaactccaatcaaataattttataaagttagtgtatcactttgta
atcataaaaacaacaataaagctacttaaatatagatttataaaaaacgttggcgaaaacgttggcgattcgttggc
gattgaaaaaccccttaaacccttgagccagttgggatagagcgtttttggcacaaaaattggcactcggcacttaa
tggggggtcgtagtacggaagcaaaattcgcttcctttccccccatttttttccaaattccaaatttttttcaaaaa
ttttccagcgctaccgctcggcaaaattgcaagcaatttttaaaatcaaacccatgagggaatttcattccctcata
ctcccttgagcctcctccaaccgaaatagaagggcgctgcgcttattatttcattcagtcatcggctttcataatct
aacagacaacatcttcgctgcaaagccacgctacgctcaagggcttttacgctacgataacgcctgttttaacgatt
atgccgataactaaacgaaataaacgctaaaacgtctcagaaacgattttgagacgttttaataaaaaatcgcctag
tgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtc
attactggatctatcaacaggagtccaagcgagctcggtactaaaacaattcatccagtaaaatataatattttatt
ttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccctgat
cgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttactt
tgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttccgtc
tttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggccag
atcgttattcagtaagtaatccaattcggctaagcggccgtctaagctattcgtatagggacaatccgatatgtcga
tggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttcagggctttgttcatcttcatacccttcc
gagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttgg
aacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttataccggc
tgtccgtcatttttaaatataggatttcattttctcccaccagcttatataccttagcaggagacattccttccgta
tcttttacgcagcggtattcttcgatcagttttttcaattccggtgatattctcattttagccatttattatttcct
tcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttccttgc
attctaaaaccttaaatacagaaaacagccttttcaaagttgttttcaaagttggcgtataacatagtatcgacgga
gccgattttgaaaccacaattatgatagaattt3344
<210>3
<211>5528
<212>DNA
<213>It is artificial synthesized
<220>
<223>PBTS-IC sequences
<400>3
gacgtcgaattcgaggtacctgcggccgcaggatccatctagataactcacattaattgcgttgcgctcactgcccg
ctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtatt
gggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagaga
gttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttgacggcgggatataa
catgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggc
gcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgca
tggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgaga
tatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtg
acccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtct
ggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagc
ggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgct
tcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacg
gcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcgg
ttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggtt
caccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatcaaaa
tcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctttccactatccctacagtgttatgg
cttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactcaaacatcaaatcttacaaatgtagtc
tttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaaggaaatgatgacctcgtttccaccggaa
ttagcttgcatgcctaatcgccatcttccagcaggcgcaccattgcccctgtttcactatccaggttacggatatag
ttcatgacaatatttacattggtccagccaccagcttgcatgatctccggtattgaaactccagcgcgggccatatc
tcgcgcggctccgacacgggcactgtgtccagaccaggccaggtatctctgaccagagtcatccttagcgccgtaaa
tcaatcgatgagttgcttcaaaaatcccttccagggcgcgagttgatagctggctggtggcagatggcgcggcaaca
ccattttttctgacccggcaaaacaggtagttattcggatcatcagctacaccagagacggaaatccatcgctcgac
cagtttagttacccccaggctaagtgccttctctacacctgcggtgctaaccagcgttttcgttctgccaatatgga
ttaacattctcccaccgtcagtacgtgagatatctttaaccctgatcctggcaatttcggctatacgtaacagggtg
ttataagcaatccccagaaatgccagattacgtatatcctggcagcgatcgctattttccatgagtgaacgaacctg
gtcgaaatcagtgcgttcgaacgctagagcctgttttgcacgttcaccggcatcaacgttttcttttcggatccgcc
gcataaccagtgaaacagcattgctgtcacttggtcgtggcagcccggaccgacgatgaagcatgtttagctggccc
aaatgttgctggatagtttttactgccagaccgcgcgcctgaagatatagaagataatcgcgaacatcttcaggttc
tgcgggaaaccatttccggttattcaacttgcaccatgccgcccacgaccggcaaacggacagaagcattttccagg
tatgctcagaaaacgcctggcgatccctgaacatgtccatcaggttcttgcgaacctcatcactcgttgcatcgacc
ggtaatgcaggcaaattttggtgtacggtcagtaaattggacatacctgcttcctccttaagcttaattgttatccg
ctcacaattccacacattatgccacaccttgtagataaagtcaacaactttttgcaaaatttttcaggaattttagc
agaggttgttctggatgtagaacaaaacatctttccgctcttgtgctgttaggatatctttcttggaagctaggtag
gcaagggctacctcaaataaatcttcttcagggtgcgctatttttaaggtgcctagtgaggtcttgaccacttcacc
cataatttcagtgccgaatagtctggactgggctgtgtactgcagtgaagcttcagggcccgatcgatgccgccgct
taattaattaatccagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtt
tgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctagtgtcattttatttcccccgtttcagca
tcaagaacctttgcataacttgctctatatccacactgataattgccctcaaaccataatctaaaggcgctagagtt
tgttgaaacaatatcttttacatcattcgtatttaaaattccaaactccgctcccctaaggcgaataaaagccatta
aatcttttgtatttaccaaattatagtcatccactatatctaagagtaaattcttcaattctcttttttggctttca
tcaagtgttatatagcggtcaatatcaaaatcattaatgttcaaaatatcttttttgtcgtatatatgtttattctt
agcaatagcgtcctttgattcatgagtcaaatattcataagaacctttgatataatcaagtatctcaacatgagcaa
ctgaactattccccaattttcgcttaatcttgttcctaacgctttctattgttacaggatttcgtgcaatatatata
acgtgatagtgtggttttttatagtgctttccatttcgtataacatcactactattccatgtatctttatctttttt
ttcgtccatatcgtgtaaaggactgacagccatagatacgcccaaactctctaatttttccttccaatcattaggaa
ttgagtcaggatataataaaaatccaaaatttctagctttagtatttttaatagccatgatataattaccttatcaa
aaacaagtagcgaaaactcgtatccttctaaaaacgcgagctttcgcttattttttttgttctgattcctttcttgc
atattcttctatagctaacgccgcaaccgcagattttgaaaaacctttttgtttcgccatatctgttaattttttat
cttgctcttttgtcagagaaatcataactctttttttcgattctgaaatcaccatttaaaaaactccaatcaaataa
ttttataaagttagtgtatcactttgtaatcataaaaacaacaataaagctacttaaatatagatttataaaaaacg
ttggcgaaaacgttggcgattcgttggcgattgaaaaaccccttaaacccttgagccagttgggatagagcgttttt
ggcacaaaaattggcactcggcacttaatggggggtcgtagtacggaagcaaaattcgcttcctttccccccatttt
tttccaaattccaaatttttttcaaaaattttccagcgctaccgctcggcaaaattgcaagcaatttttaaaatcaa
acccatgagggaatttcattccctcatactcccttgagcctcctccaaccgaaatagaagggcgctgcgcttattat
ttcattcagtcatcggctttcataatctaacagacaacatcttcgctgcaaagccacgctacgctcaagggctttta
cgctacgataacgcctgttttaacgattatgccgataactaaacgaaataaacgctaaaacgtctcagaaacgattt
tgagacgttttaataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttct
gaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctcggtactaaaacaa
ttcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacat
actgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgctt
ctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaa
gacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttctt
cccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggccgtctaagcta
ttcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttc
agggctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccat
catgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttcc
acatcataggtggtccctttataccggctgtccgtcatttttaaatataggatttcattttctcccaccagcttata
taccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatcagttttttcaattccggtgata
ttctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataaca
agacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgttttcaa
agttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgatagaattt5528
Sequence table
SEQUENCE LISTING
<110>Chengdu Mei Yide Bioisystech Co., Ltd
<120>A kind of method for transforming bacillus gene group
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 2847
<212> DNA
<213>It is artificial synthesized
<220>
<223>PBTS sequences
<400> 1
gacgtcgaattcgaggtacctgcggccgcaggatccatctagacgctcgagagctgcagtgaagcttcagggc
ccgatcgatgccgccgcttaattaattaatccagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcct
ttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctagtgtcatttt
atttcccccgtttcagcatcaagaacctttgcataacttgctctatatccacactgataattgccctcaaaccataa
tctaaaggcgctagagtttgttgaaacaatatcttttacatcattcgtatttaaaattccaaactccgctcccctaa
ggcgaataaaagccattaaatcttttgtatttaccaaattatagtcatccactatatctaagagtaaattcttcaat
tctcttttttggctttcatcaagtgttatatagcggtcaatatcaaaatcattaatgttcaaaatatcttttttgtc
gtatatatgtttattcttagcaatagcgtcctttgattcatgagtcaaatattcataagaacctttgatataatcaa
gtatctcaacatgagcaactgaactattccccaattttcgcttaatcttgttcctaacgctttctattgttacagga
tttcgtgcaatatatataacgtgatagtgtggttttttatagtgctttccatttcgtataacatcactactattcca
tgtatctttatcttttttttcgtccatatcgtgtaaaggactgacagccatagatacgcccaaactctctaattttt
ccttccaatcattaggaattgagtcaggatataataaaaatccaaaatttctagctttagtatttttaatagccatg
atataattaccttatcaaaaacaagtagcgaaaactcgtatccttctaaaaacgcgagctttcgcttattttttttg
ttctgattcctttcttgcatattcttctatagctaacgccgcaaccgcagattttgaaaaacctttttgtttcgcca
tatctgttaattttttatcttgctcttttgtcagagaaatcataactctttttttcgattctgaaatcaccatttaa
aaaactccaatcaaataattttataaagttagtgtatcactttgtaatcataaaaacaacaataaagctacttaaat
atagatttataaaaaacgttggcgaaaacgttggcgattcgttggcgattgaaaaaccccttaaacccttgagccag
ttgggatagagcgtttttggcacaaaaattggcactcggcacttaatggggggtcgtagtacggaagcaaaattcgc
ttcctttccccccatttttttccaaattccaaatttttttcaaaaattttccagcgctaccgctcggcaaaattgca
agcaatttttaaaatcaaacccatgagggaatttcattccctcatactcccttgagcctcctccaaccgaaatagaa
gggcgctgcgcttattatttcattcagtcatcggctttcataatctaacagacaacatcttcgctgcaaagccacgc
tacgctcaagggcttttacgctacgataacgcctgttttaacgattatgccgataactaaacgaaataaacgctaaa
acgtctcagaaacgattttgagacgttttaataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccg
gcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagcg
agctcggtactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagt
caaaaaatagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccac
ttgtccgccctgccgcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcc
caggtcgccgtgggaaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctt
taaatggagtgtcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggct
aagcggccgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcata
cagctcgatagtcttttcagggctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatga
gcagattgctccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatc
atgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggatttcatt
ttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatcagtt
ttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagatacccc
aagaagctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaacagcct
tttcaaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgatagaat
tt 2847
<210> 2
<211> 3344
<212> DNA
<213>It is artificial synthesized
<220>
<223>PBTS-Z sequences
<400> 2
gacgtcgaattcgaggtacctgcggccgcaggatcctaccgttcgtatagcatacattatacgaagttatctt
gatatggctttttatatgtgttactctacatacagaaaggaggaactaaatatggccaagttgaccagtgccgttcc
ggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtgg
aggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggac
aacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaactt
ccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccgg
ccggcaactgcgtgcacttcgtggccgaggagcaggactgaataacttcgtatagcatacattatacgaacggtaGt
ctagacgctcgagagctgcagtgaagcttcagggcccgatcgatgccgccgcttaattaattaatccagaggcatca
aataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagta
ggacaaatccgccgccctagacctagtgtcattttatttcccccgtttcagcatcaagaacctttgcataacttgct
ctatatccacactgataattgccctcaaaccataatctaaaggcgctagagtttgttgaaacaatatcttttacatc
attcgtatttaaaattccaaactccgctcccctaaggcgaataaaagccattaaatcttttgtatttaccaaattat
agtcatccactatatctaagagtaaattcttcaattctcttttttggctttcatcaagtgttatatagcggtcaata
tcaaaatcattaatgttcaaaatatcttttttgtcgtatatatgtttattcttagcaatagcgtcctttgattcatg
agtcaaatattcataagaacctttgatataatcaagtatctcaacatgagcaactgaactattccccaattttcgct
taatcttgttcctaacgctttctattgttacaggatttcgtgcaatatatataacgtgatagtgtggttttttatag
tgctttccatttcgtataacatcactactattccatgtatctttatcttttttttcgtccatatcgtgtaaaggact
gacagccatagatacgcccaaactctctaatttttccttccaatcattaggaattgagtcaggatataataaaaatc
caaaatttctagctttagtatttttaatagccatgatataattaccttatcaaaaacaagtagcgaaaactcgtatc
cttctaaaaacgcgagctttcgcttattttttttgttctgattcctttcttgcatattcttctatagctaacgccgc
aaccgcagattttgaaaaacctttttgtttcgccatatctgttaattttttatcttgctcttttgtcagagaaatca
taactctttttttcgattctgaaatcaccatttaaaaaactccaatcaaataattttataaagttagtgtatcactt
tgtaatcataaaaacaacaataaagctacttaaatatagatttataaaaaacgttggcgaaaacgttggcgattcgt
tggcgattgaaaaaccccttaaacccttgagccagttgggatagagcgtttttggcacaaaaattggcactcggcac
ttaatggggggtcgtagtacggaagcaaaattcgcttcctttccccccatttttttccaaattccaaatttttttca
aaaattttccagcgctaccgctcggcaaaattgcaagcaatttttaaaatcaaacccatgagggaatttcattccct
catactcccttgagcctcctccaaccgaaatagaagggcgctgcgcttattatttcattcagtcatcggctttcata
atctaacagacaacatcttcgctgcaaagccacgctacgctcaagggcttttacgctacgataacgcctgttttaac
gattatgccgataactaaacgaaataaacgctaaaacgtctcagaaacgattttgagacgttttaataaaaaatcgc
ctagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctga
ggtcattactggatctatcaacaggagtccaagcgagctcggtactaaaacaattcatccagtaaaatataatattt
tattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccc
tgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccactt
actttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttc
cgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcgg
ccagatcgttattcagtaagtaatccaattcggctaagcggccgtctaagctattcgtatagggacaatccgatatg
tcgatggagtgaaagagcctgatgcactccgcatacagctcgatagtcttttcagggctttgttcatcttcataccc
ttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacct
ttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttatac
cggctgtccgtcatttttaaatataggatttcattttctcccaccagcttatataccttagcaggagacattccttc
cgtatcttttacgcagcggtattcttcgatcagttttttcaattccggtgatattctcattttagccatttattatt
tccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttcc
ttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgttttcaaagttggcgtataacatagtatcga
cggagccgattttgaaaccacaattatgatagaattt 3344
<210> 3
<211> 5528
<212> DNA
<213>It is artificial synthesized
<220>
<223>PBTS-IC sequences
<400> 3
gacgtcgaattcgaggtacctgcggccgcaggatccatctagataactcacattaattgcgttgcgctcactg
cccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcg
tattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctga
gagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttgacggcgggat
ataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaa
tggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatt
tgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagt
gagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgct
ggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggt
gtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatc
cagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgc
cgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgc
gacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccac
gcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcct
ggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatc
aaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctttccactatccctacagtgtt
atggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactcaaacatcaaatcttacaaatgt
agtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaaggaaatgatgacctcgtttccacc
ggaattagcttgcatgcctaatcgccatcttccagcaggcgcaccattgcccctgtttcactatccaggttacggat
atagttcatgacaatatttacattggtccagccaccagcttgcatgatctccggtattgaaactccagcgcgggcca
tatctcgcgcggctccgacacgggcactgtgtccagaccaggccaggtatctctgaccagagtcatccttagcgccg
taaatcaatcgatgagttgcttcaaaaatcccttccagggcgcgagttgatagctggctggtggcagatggcgcggc
aacaccattttttctgacccggcaaaacaggtagttattcggatcatcagctacaccagagacggaaatccatcgct
cgaccagtttagttacccccaggctaagtgccttctctacacctgcggtgctaaccagcgttttcgttctgccaata
tggattaacattctcccaccgtcagtacgtgagatatctttaaccctgatcctggcaatttcggctatacgtaacag
ggtgttataagcaatccccagaaatgccagattacgtatatcctggcagcgatcgctattttccatgagtgaacgaa
cctggtcgaaatcagtgcgttcgaacgctagagcctgttttgcacgttcaccggcatcaacgttttcttttcggatc
cgccgcataaccagtgaaacagcattgctgtcacttggtcgtggcagcccggaccgacgatgaagcatgtttagctg
gcccaaatgttgctggatagtttttactgccagaccgcgcgcctgaagatatagaagataatcgcgaacatcttcag
gttctgcgggaaaccatttccggttattcaacttgcaccatgccgcccacgaccggcaaacggacagaagcattttc
caggtatgctcagaaaacgcctggcgatccctgaacatgtccatcaggttcttgcgaacctcatcactcgttgcatc
gaccggtaatgcaggcaaattttggtgtacggtcagtaaattggacatacctgcttcctccttaagcttaattgtta
tccgctcacaattccacacattatgccacaccttgtagataaagtcaacaactttttgcaaaatttttcaggaattt
tagcagaggttgttctggatgtagaacaaaacatctttccgctcttgtgctgttaggatatctttcttggaagctag
gtaggcaagggctacctcaaataaatcttcttcagggtgcgctatttttaaggtgcctagtgaggtcttgaccactt
cacccataatttcagtgccgaatagtctggactgggctgtgtactgcagtgaagcttcagggcccgatcgatgccgc
cgcttaattaattaatccagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgt
tgtttgtcggtgaacgctctcctgagtaggacaaatccgccgccctagacctagtgtcattttatttcccccgtttc
agcatcaagaacctttgcataacttgctctatatccacactgataattgccctcaaaccataatctaaaggcgctag
agtttgttgaaacaatatcttttacatcattcgtatttaaaattccaaactccgctcccctaaggcgaataaaagcc
attaaatcttttgtatttaccaaattatagtcatccactatatctaagagtaaattcttcaattctcttttttggct
ttcatcaagtgttatatagcggtcaatatcaaaatcattaatgttcaaaatatcttttttgtcgtatatatgtttat
tcttagcaatagcgtcctttgattcatgagtcaaatattcataagaacctttgatataatcaagtatctcaacatga
gcaactgaactattccccaattttcgcttaatcttgttcctaacgctttctattgttacaggatttcgtgcaatata
tataacgtgatagtgtggttttttatagtgctttccatttcgtataacatcactactattccatgtatctttatctt
ttttttcgtccatatcgtgtaaaggactgacagccatagatacgcccaaactctctaatttttccttccaatcatta
ggaattgagtcaggatataataaaaatccaaaatttctagctttagtatttttaatagccatgatataattacctta
tcaaaaacaagtagcgaaaactcgtatccttctaaaaacgcgagctttcgcttattttttttgttctgattcctttc
ttgcatattcttctatagctaacgccgcaaccgcagattttgaaaaacctttttgtttcgccatatctgttaatttt
ttatcttgctcttttgtcagagaaatcataactctttttttcgattctgaaatcaccatttaaaaaactccaatcaa
ataattttataaagttagtgtatcactttgtaatcataaaaacaacaataaagctacttaaatatagatttataaaa
aacgttggcgaaaacgttggcgattcgttggcgattgaaaaaccccttaaacccttgagccagttgggatagagcgt
ttttggcacaaaaattggcactcggcacttaatggggggtcgtagtacggaagcaaaattcgcttcctttcccccca
tttttttccaaattccaaatttttttcaaaaattttccagcgctaccgctcggcaaaattgcaagcaatttttaaaa
tcaaacccatgagggaatttcattccctcatactcccttgagcctcctccaaccgaaatagaagggcgctgcgctta
ttatttcattcagtcatcggctttcataatctaacagacaacatcttcgctgcaaagccacgctacgctcaagggct
tttacgctacgataacgcctgttttaacgattatgccgataactaaacgaaataaacgctaaaacgtctcagaaacg
attttgagacgttttaataaaaaatcgcctagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcg
ttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctcggtactaaa
acaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcg
acatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgcc
gcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtggg
aaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtct
tcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggccgtctaa
gctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgatagtct
tttcagggctttgttcatcttcatacccttccgagcaaaggacgccatcggcctcactcatgagcagattgctccag
ccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccg
ttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggatttcattttctcccaccagct
tatataccttagcaggagacattccttccgtatcttttacgcagcggtattcttcgatcagttttttcaattccggt
gatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattat
aacaagacgaactccaattcactgttccttgcattctaaaaccttaaatacagaaaacagccttttcaaagttgttt
tcaaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgatagaattt 5528
Claims (7)
1. it is a kind of transform bacillus gene group method, it is characterised in that：Comprise the following steps：
(1) EcoR I and the Apa I site in pBAV1K-T5-GFP plasmids insert MCS fragment, and by synonymous
Mutation, deletes NdeI restriction enzyme sites therein, obtains plasmid pBTS；
(2) restriction enzyme site in the left end of MCS inserts the homologous recombination fragment A of 500-800bp, right side restriction enzyme site
The homologous recombination fragment B of 500-800bp is inserted, middle insertion mutation fragment, purpose fragment or any fragment is not inserted into, both two
The fragment of transformation is needed between homologous recombination region, plasmid pBTS-X is obtained；
(3) pBTS-X is transferred in bacillus by electricity conversion, obtains positive transformants bacterial strain；
(4) into being cultivated in fluid nutrient medium, cultivation temperature is 45 DEG C to the positive transformants bacterium colony in picking (3), during culture
Between be more than 24h；
(5) bacterium solution 100ul -500ul in step (4) is taken, is applied on the LB flat boards containing 30mg/L kanamycins, cultivation temperature
It is 45 DEG C, carries out incubated overnight；
(6) bacterium colony in picking step (5) is into being cultivated in fluid nutrient medium, every 8-16h passages once, until screening
To nonreactive bacterial strain；
(7) take the bacterium solution in step (6) and be diluted coated plate, dilution 105-106Times, incubated overnight；
(8) bacterium colony in picking step (7) carries out Resistance Identification, until obtaining 5-10 plants of nonreactive bacterial strain；
(9) nonreactive bacterial strain is inoculated into fluid nutrient medium, incubated overnight, extracts bacterial genomes, enter performing PCR identification；It is positive
Bacterial strain proceeds sequencing identification, and sequencing identification result obtains final product Bacillus strain for positive strain.
2. it is according to claim 1 it is a kind of transform bacillus gene group method, it is characterised in that：The step (3)
Middle bacillus is bacillus subtilis 168, Z12 bacterial strains, bacillus amyloliquefaciens, bacillus pumilus, bacillus licheniformis
Or other pBTS plasmids have the gram-positive bacteria of temperature sensitive copy characteristic.
3. it is according to claim 2 it is a kind of transform bacillus gene group method, it is characterised in that：The gram sun
Property bacterium be streptococcus pneumonia, Lactococcus lactis or series bacillus bacterial strain.
4. it is according to claim 1 it is a kind of transform bacillus gene group method, it is characterised in that：The pBTS nucleosides
Acid sequence is as shown in SEQ ID NO.1.
5. it is according to claim 1 it is a kind of transform bacillus gene group method, it is characterised in that：Including following step
Suddenly：(1) the MCS left-hand end in pBTS inserts the homologous recombination fragment A of 400-800bp, right-hand end insertion 400-
The homologous recombination fragment B of 800bp, middle insertion mutation fragment or purpose fragment or is not inserted into fragment, then in the transformation of B with centre
Insertion has H chloramphenicol resistance fragments between fragment or A and the transformation fragment of centre, and cre weights are contained at H chloramphenicol resistance fragments two ends
Lox71 the and lox72 fragments of group enzyme identification, the plasmid is named as pBTS-X-Z；
(2) pBTS-X-Z plasmids are converted by electricity conversion and is entered in bacillus, obtain positive transformants bacterial strain；
(3) into being cultivated in fluid nutrient medium, cultivation temperature is 45 DEG C to the positive transformants bacterium colony in picking step (2), training
The time of supporting is more than 24h；
(4) bacterium solution 100ul -500ul in (3) is taken in step, is applied on the flat board containing kanamycins or H mycins, culture temperature
45 DEG C of degree, incubated overnight；
(5) bacterium colony in picking (4) enters and is cultivated in the LB liquid medium containing H chloramphenicol resistances, every 8-16h passages
Once, only there is the bacterium colony of H chloramphenicol resistances until screening；
(6) take the bacterium solution in (5) and be diluted coated plate, flat board contains H chloramphenicol resistances, dilution 105-106Times, incubated overnight；
(7) bacterium colony in picking (6) carries out Resistance Identification, only has H chloramphenicol resistances until obtaining 5-10 plants, and without card, that is mould
The bacterial strain of plain resistance；
(8) monoclonal antibody inoculation is taken in fluid nutrient medium, incubated overnight extracts bacterial genomes, enter performing PCR identification, it is positive
Bacterial strain proceeds sequencing identification, and sequencing identification is that positive bacterial strain enters next step；
(9) conversion pBTS-IC plasmids enter the monoclonal antibody bacterial strain that the positive is accredited as in (8), and IC is the regulation and control cre restructuring of lactose operator
The fragment of expression of enzymes, cre recombinases can characteristic identification lox71 and lox66 sites, recombinated, being left in genome can not
The loxP sites recognized by cre recombinases, the lower fragment of cutting contains the lox72 sites for easily recognizing；
(10) the positive transformants bacterial strain in picking (9), connects bacterium and is cultivated, using IPTG induced expressions or leakage expression
Cre recombinates cleavage H chloramphenicol resistance fragments；
(11) bacterium solution in (10) is taken, is inoculated into fluid nutrient medium, cultivation temperature is 45 DEG C, and incubation time is more than 24h；
(12) bacterium solution of culture in (11), dilution 10 are taken5-106To on the flat board of nonreactive, cultivation temperature is 45 DEG C to coated plate, is overnight trained
Support；
(13) single bacterium colony in (12) is taken, Resistance Identification is carried out, until screens 2-5 plants of nonreactive bacterial strain；
(14) nonreactive bacterial strain in (13) is taken, is inoculated into culture medium, incubated overnight extracts genome, enter performing PCR identification, sun
Property bacterial strain carry out sequencing identification again, be still accredited as the positive bacterial strain be complete genome manipulation bacterial strain.
6. it is according to claim 5 it is a kind of transform bacillus gene group method, it is characterised in that：The H mycins are
Any in bleomycin, tetracycline, chloramphenicol and spectinomycin.
7. it is according to claim 1 it is a kind of transform bacillus gene group method, it is characterised in that：The pBTS-IC
Nucleotide sequence is as shown in SEQ ID NO.3.
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| CN107779465A (en) * | 2017-10-30 | 2018-03-09 | 成都美溢德生物技术有限公司 | A kind of method for reducing fermentation of bacillus liquid viscosity |
| CN109337855A (en) * | 2018-11-20 | 2019-02-15 | 成都美溢德生物技术有限公司 | A kind of efficient method for reducing fermentation of bacillus liquid viscosity |
| CN112239742A (en) * | 2020-08-24 | 2021-01-19 | 天津科技大学 | Bacillus licheniformis for producing alkaline protease and application thereof |
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| CN102021164A (en) * | 2010-11-09 | 2011-04-20 | 南京农业大学 | Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene |
| CN102311970A (en) * | 2011-08-05 | 2012-01-11 | 浙江大学 | Expression cassette mazF-cassette for expressing escherichia coli toxin protein mazF gene and construction method and application thereof |
| CN104388460A (en) * | 2014-11-26 | 2015-03-04 | 成都美溢德生物技术有限公司 | Method for transforming bacillus |
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| CN107779465B (en) * | 2017-10-30 | 2021-02-26 | 成都美溢德生物技术有限公司 | Method for reducing viscosity of bacillus fermentation liquid |
| CN109337855A (en) * | 2018-11-20 | 2019-02-15 | 成都美溢德生物技术有限公司 | A kind of efficient method for reducing fermentation of bacillus liquid viscosity |
| CN112239742A (en) * | 2020-08-24 | 2021-01-19 | 天津科技大学 | Bacillus licheniformis for producing alkaline protease and application thereof |
| CN112239742B (en) * | 2020-08-24 | 2022-10-18 | 天津科技大学 | Bacillus licheniformis for producing alkaline protease and application thereof |
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